Fibromyalgia is a persistent muscle pain that can be accompanied by severe fatigue, insomnia, diarrhea, abdominal bloating, bladder irritation and headache. The criteria for a diagnosis of fibromyalgia may include widespread pain throughout the body accompanied by tenderness in 11 of 18 specific tender points (FIG. 1). Tenderness is determined by applying firm pressure over each designated area.
The underlying pathophysiology and pathology of fibromyalgia is not well understood. Radiographic and histological examinations of regions associated with tenderness reveal no abnormalities. Blood chemistries, CBC, erythrocyte sedimentation rate (ESR), as well as other immunologic manifestations commonly known for other diseases (i.e. autoantibodies in Lupus), are normally negative unless there is another underlying disorder.
The source of the pain appears to be somewhat unclear. Nociceptors are present in the interstitial space between muscle fibers, in particular, on blood vessels. Studies have reported intramuscular microcirculation abnormalities as well as a decrease in energy-rich phosphates in fibromyalgia musculature, Raj et al, Pain Digest, 8(6), 357-363 (1998)). These abnormalities may be important for producing muscle associated pain since 1) impaired microcirculation results in insufficient delivery of oxygen to localized muscle regions which results in sub-optimal working capacity of the musculature which may lead to exhaustion of some motor units; and 2) reduced energy-rich phosphates (ATP and phosphorylcreatine) means that demands of working muscles are not met by the energy supply, which may cause localized muscular strain, weakness, fatigue and pain.
Fibromyalgia tender points are characterized by allodynia (axe2x80xa2state where a normally non-painful stimulus elicits a painful perception). Muscular dysfunction, either mechanical or metabolic, can lead to a state of sensitization of the nociceptive sensory inputs into the spinal cord altering the neurochemical balance important for nociceptive control. The process of nociception may be accomplished by a controlled release of various pro-nociceptive and anti-nociceptive agents in the nervous system. These agents include excitatory amino acids, neuropeptides, biogenic amines, nitric oxide, and prostaglandins. One important pro-nociceptive mediator is the neuropeptide substance P, which is found to be consistently elevated in the cerebrospinal fluid of fibromyalgia patients. Substance P may be released by sensory afferents arising from the muscle into the dorsal horn of the spinal cord to interact with neurokinin-1 receptors. Activation of spinal neurons by substance P prepares the neurons for an inceptive pain signal, thereby facilitating nociceptive perception. Injection of substance P into animals causes allodynia by increasing the number of afferent neurons that are activated (e.g. discharge one or more action potentials) in response to a certain nociceptive stimulus and reducing the voltage threshold needed for their activation.
A recent finding of elevated nerve growth factor levels in cerebral spinal fluid of patients may exacerbate the condition by increasing the development of substance P-containing sensory neurons, which either contribute to or accentuate the painful symptoms brought about by an elevated level of substance P. Further, levels of anti-nociceptive substances such as the neuropeptide met-enkephalin and biogenic amine serotonin are found to be significantly reduced in fibromyalgia patients"" cerebral spinal fluid.
Fibromyalgia tends to be chronic and often occurs after a stressful event suffered by the patient, either physical or psychological. Current treatments involve massages, exercise, changes in diet and anti-depressant medication. All of these forms of treatment are inadequate only providing some benefit to a small subset of patients.
What is needed are new effective methods to treat fibromyalgia, including pain associated with fibromyalgia. The present invention provides methods to treat the symptoms (including pain) of fibromyalgia by the injection of a Clostridial toxin, for example, a botulinum toxin, into a patient at a location that is not at or near a site where the patient perceives the pain to originate. It is hypothesized that botulinum toxin may interfere with the central pain pathway through routes not traditionally associated with the action of this neurotoxin.
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
Botulinum toxin type A (xe2x80x9cBoNT/Axe2x80x9d) is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) serotype A is a LD50 in mice. One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18-20 grams each. Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G each of which is distinguished by neutralization with serotype-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that BoNt/A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin serotype B (BoNT/B). Additionally, botulinum toxin type B (xe2x80x9cBoNt/Bxe2x80x9d) has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for BoNt/A. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. BoNt/A has been approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus, hemifacial spasm and cervical dystonia. Additionally a botulinum toxin type B has been approved by the FDA for the treatment of cervical dystonia.
Non-serotype A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to BoNt/A. Clinical effects of peripheral intramuscular BoNt/A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of BoNt/A averages about three months.
Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum serotypes A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. BoNT/B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin serotype C1 (BoNT/C1) has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.
Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the H chain and a cell surface receptor; the receptor is thought to be different for each serotype of botulinum toxin and for tetanus toxin. The carboxyl end segment of the H chain, Hc, appears to be important for targeting of the toxin to the cell surface.
In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This last step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin then translocates through the endosomal membrane into the cytosol.
The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the H and L chain. The entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin/B/D,/F, and/G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the BoNt/A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. BoNT/B and C1 are apparently produced as only a 500 kD complex. BoNT/D is produced as both 300 kD and 500 kD complexes. Finally, BoNT/E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
BoNt/A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the BoNt/B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the BoNt/B toxin is likely to be inactive, possibly accounting for the known significantly lower potency of BoNt/B as compared to BoNt/A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy. Additionally, it is known that BoNt/B has, upon intramuscular injection, a shorter duration of activity and is also less potent than BoNt/A at the same dose level.
It has been reported (as exemplary examples) that BoNt/A has been used clinically as follows:
(1) about 75-125 units of BOTOX(copyright)1 per intramuscular injection (multiple muscles) to treat cervical dystonia;
(2) 5-10 units of BOTOX(copyright) per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);
(3) about 30-80 units of BOTOX(copyright) to treat constipation by intrasphincter injection of the puborectalis muscle;
(4) about 1-5 units per muscle of intramuscularly injected BOTOX(copyright) to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.
(5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 units of BOTOX(copyright), the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).
(6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX(copyright) into five different upper limb flexor muscles, as follows:
(a) flexor digitorum profundus: 7.5 U to 30 U
(b) flexor digitorum sublimus: 7.5 U to 30 U
(c) flexor carpi ulnaris: 10 U to 40 U
(d) flexor carpi radialis: 15 U to 60 U
(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX(copyright) by intramuscular injection at each treatment session.
1Available from Allergan, Inc., of Irvine, Calif. under the tradename BOTOX(copyright). 
The tetanus neurotoxin acts mainly in the central nervous system, while botulinum neurotoxin acts at the neuromuscular junction; both act by inhibiting acetylcholine release from the axon of the affected neuron into the synapse, resulting in paralysis. The effect of intoxication on the affected neuron is long-lasting and until recently has been thought to be irreversible. The tetanus neurotoxin is known to exist in one immunologically distinct serotype.
Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic and most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of the heart by the vagal nerve.
The efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system. The preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers, extending from the cell body, synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the synapses between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic. The nicotinic receptors are also present in many membranes of skeletal muscle fibers at the neuromuscular junction.
Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane. A wide variety of non-neuronal secretory cells, such as, adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and insulin, respectively, from large dense-core vesicles. The PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development. Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell. Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
In accordance with the present invention, there are provided methods for treating fibromyalgia. These methods may include administering locally a therapeutically effective amount of a Clostridial toxin to a peripheral location on a body of a patient afflicted with fibromyalgia. The peripheral location of local administration is not a locus of pain. For example, the peripheral location may be on the body of a patient about one centimeter or more from the locus of pain. In one embodiment, the locus of pain is a fibromyalgia tender point.
In one embodiment of the present invention, a dermatome may include both the locus of pain, for example, where a pain associated with fibromyalgia is perceived by the patient to originate, and the peripheral location where the therapeutically effective amount of a Clostridial toxin is administered.
In another embodiment, the peripheral location where an effective amount of Clostridial neurotoxin is administered is in the head of the patient. For example, the neurotoxin may be administered to the facial area and/or cranial area of the head. In this embodiment, where an effective amount of Clostridial neurotoxin is administered in the head of the patient, the locus of pain to be treated is not in the head. For example, the locus of pain may be at a fibromyalgia tender point.
Further in accordance with the present invention, there are provided methods for treating pain which may include administering locally a therapeutically effective amount of a Clostridial toxin to a peripheral location on a body of a patient. The site of local administration is other than a locus of pain. A dermatome may include both a locus of pain and a site of local administration. In one embodiment, the locus of pain may be at a fibromyalgia tender point. In one embodiment, a patient has at least eleven loci of pain.
Further in accordance with the present invention, there are provided methods for treating fibromyalgia. These methods may include administering locally a therapeutically effective amount of a Clostridial toxin to a dermatome of a patient afflicted with fibromyalgia. This dermatome may substantially include a locus of pain. The local administering is not at a locus of pain. In one embodiment of the invention, the locus of pain is at one or more fibromyalgia tender points.
Methods for treating pain are within the scope of the invention. These methods may administering locally a therapeutically effective amount of a Clostridial toxin at a location in a dermatome other than a locus of pain. This dermatome also includes a locus of pain. The locus of pain may be at a fibromyalgia tender point.
Further in accordance with the present invention, there are provided methods for treating fibromyalgia pain. These methods include a step of administering a therapeutically effective amount of botulinum toxin type A to a dermatome. This dermatome may include a site where the pain is perceived by the patient to originate.
Still further in accordance with the present invention, there are provided methods for treating pain which is perceived to originate at fibromyalgia tender point. These methods include a step of administering a therapeutically effective amount of botulinum toxin type A to a location in a dermatome other than where the pain is perceived to originate. This dermatome includes the site where the pain is perceived to originate.
A toxin used in accordance with the present invention may be botulinum toxin type A, B, C1, C2, D, E, F, G or fragments of these toxins or derivatives of these toxins. In one embodiment of the present invention, the toxin may be a mixture or combination of these botulinum toxins.
The location where the therapeutically effective amount of a toxin is administered and the site where a pain is perceived by the patient to originate may have neuronal processes that project from the same spinal sensory nerve root.
Further in accordance with the present invention, a toxin may be administered, for example, subcutaneously or intramuscularly. Further, the toxin may be administered with a needle or by needleless injection.
In one embodiment of the present invention, there is provided a method for treating fibromyalgia which may include administering locally a therapeutically effective amount of a botulinum toxin type A to a dermatome of a patient afflicted with fibromyalgia. The dermatome substantially encompasses a locus of pain and the locus of pain is at a fibromyalgia tender point. The local administration is not at the locus of pain.
In one embodiment of the present invention, there is provided a method for treating pain which may include administering locally a therapeutically effective amount of a botulinum toxin type A to a dermatome of a patient. The patient has a locus of pain which is at a fibromyalgia tender point, wherein the dermatome substantially encompasses the locus of pain, and wherein the local administration is not to the locus of pain.
Any and all features described herein and combinations of such features are included within the scope of the invention provided that such features of any such combination are not mutually exclusive.
These and other aspects and advantages of the present invention are apparent in the following detailed description and claims.
xe2x80x9cAdministering locallyxe2x80x9d means administering a pharmaceutical by a non-systemic route, such as by intramuscular or subcutaneous injection or implantation of a suitable implant. Thus, for example, oral and intravenous routes of administering are excluded from the scope of xe2x80x9cadministering locally.xe2x80x9d
xe2x80x9cClostridial toxinxe2x80x9d means a toxin produced naturally by the genus of bacteria Clostridium. For example, Clostidial toxins include, but are not limited to, botulinum toxins, tetanus toxins, difficile toxins and butyricum toxins. A Clostridial toxin can also be made by known recombinant means by a non-Clostridial bacterium. 
xe2x80x9cCombinationxe2x80x9d means an ordered sequence of elements. For example, a combination of botulinum toxins may mean administration of botulinum toxin E, followed by administration of botulinum toxin type A, followed by administration of botulinum toxin type B. This is opposed to a xe2x80x9cmixturexe2x80x9d where, for example, different toxin types are combined prior to administration.
xe2x80x9cDermatomexe2x80x9d means a segment of a human body innervated by a single dorsal root. Dermatomes follow a highly regular pattern on the body (FIG. 3) and are categorized into four major regions, the cervical (C), the thoracic (T), the lumber(L) and the sacral (S) regions (FIG. 3). A dermatome may substantially encompass a locus of pain.
xe2x80x9cDerivativexe2x80x9d means a chemical entity which is slightly different from a parent chemical entity but which still has a biological effect similar, or substantially similar, to the biological effect of the chemical entity. The biological effect of the derivative may be substantially the same or better than that of the parent. For example, a derivative neurotoxin component may have one or more amino acid substitutions, amino acid modifications, amino acid deletions and/or amino acid additions. An amino acid substitution may be conservative or non-conservative, as is well understood in the art. In addition, derivatives of neurotoxin components may include neurotoxin components that have modified amino acid side chains, as is well known in the art.
An example of a derivative neurotoxin component may comprise a light chain of a botulinum toxin having one or more amino acids substituted, modified, deleted and/or added. For example, a derivative light chain may have the same or an increased ability to prevent exocytosis compared to the native light chain. For example, preventing the release of neurotransmitter vesicles. Additionally, the biological effect of a derivative may be decreased compared to the parent chemical entity. For example, a derivative light chain of a botulinum toxin type A having an amino acid sequence removed may have a shorter biological persistence than that of the parent (or native) botulinum toxin type A light chain.
xe2x80x9cFibromylagia tender pointxe2x80x9d or xe2x80x9ctender pointxe2x80x9d means an area of a human body which when firm even, pressure is applied pain may result. Elicitation of pain from 11 of 18 fibromyalgia tender points, by application of firm even pressure, results in a diagnosis of fibromyalgia. FIG. 1 shows the location of the tender points. They are: a) occiput, suboccipital muscle insertions; b) trapezius, midpoint of the upper border; c) supraspinatus, above the medial border of the scapular spine; d) gluteal, upper and outer quadrant of buttocks; e) greater trochanter, posterior to the trochanteric prominence; f) low cervical, anterior aspects of the intertransverse spaces; g) second rib, second costochondral junctions; h) lateral epicondyle, 2 centimeters distant to the epicondyles; and i) knee, medial fat pad proximal to the joint line.
xe2x80x9cFragmentxe2x80x9d means a portion of an amino acid sequence that comprises five amino acids or more of the native amino acid sequence up to a size of minus at least one amino acid from the native sequence. For example, a fragment of a botulinum toxin type A light chain comprises five or more amino acids of the amino acid sequence of the native botulinum toxin type A light chain up to a size of minus one amino acid from the native light chain.
xe2x80x9cLocus of painxe2x80x9d means a site or location on a body of a patient where the patient perceives that a pain is emanating from and/or a site or location on a body of a patient where application of pressure results in pain. For example, a locus of pain may be at a fibromyalgia tender point. Furthermore, a locus pain may have certain discrete physical boundaries. For example, a locus of pain may include a dermis area of less than about 4 cm2, 12 cm2, 40 cm2 or 60 cm2. The locus of pain may extend to tissues directly below the dermis area. For example, the locus of pain may extend about 0.5 cm, 1.0 cm, 2 cm, 4 cm or 6 cm below the dermis area.
xe2x80x9cNeedleless injectionxe2x80x9d means injecting a measurable amount of substance, for example, a carrier coated with a botulinum toxin without the use of a standard needle.
xe2x80x9cNeurotoxinxe2x80x9d means a chemical entity (i.e. a molecule) that is capable of interfering with or influencing a function of a neuron or other target cell. For example, a neurotoxin may interfere with the transmission of an electrical signal from a nerve cell to its target. The target may be, for example, another nerve cell, a tissue or an organ. The xe2x80x9cneurotoxinxe2x80x9d may be naturally occurring or synthetic.
xe2x80x9cPeripheral locationxe2x80x9d means a site on or in the periphery of a mammals"" body, such as in or under the skin or a skeletal muscle. Peripheral location excludes a site within the viscera or in the central nervous system.
xe2x80x9cSubstantiallyxe2x80x9d means largely but not entirely. For example, substantially encompassing may mean encompassing 10%, 20%, 30%, 40% or 50% or more.